Structural and thermodynamic studies of two centrin isoforms from Blastocladiella emersonii upon calcium binding

Centrins are calcium-binding proteins associated with microtubules organizing centers. Members of two divergent subfamilies of centrins were found in the aquatic fungus Blastocladiella emersonii, contrasting with the occurrence of only one member known for the better explored terrestrial fungi. BeCen1 shows greatest identity with human centrins HsCen1, HsCen2 and green algae centrin CrCenp, while BeCen3 records largest identity with human centrin HsCen3 and yeast centrin Cdc31p. Following the discovery of this unique feature, BeCen1 and BeCen3 centrins were produced to study whether these proteins had distinct features upon calcium binding. Circular dichroism showed opposite calcium binding effects on the α-helix arrangement of the secondary structure. The spectra indicated a decrease in α-helix signal for holo-BeCen1 contrasting with an increase for holo-BeCen3. In addition, only BeCen1 refolds after being de-natured. The fluorescence emission of the hydrophobic probe ANS increases for both proteins likely due to hydrophobic exposure, however, only BeCen1 presents a clear blue shift when calcium is added. ITC experiments identified four calcium binding sites for both proteins. In contrast to calcium binding to BeCen1, which is mainly endothermic, binding to BeCen3 is mainly exothermic. Light-scattering evidenced the formation of large particles in solution for BeCen1 and BeCen3 at temperatures above 30 °C and 40 °C, respectively. Atomic force microscopy confirmed the presence of supramolecular structures, which differ in the compactness and branching degree. Binding of calcium leads to different structural changes in BeCen1 and BeCen3 and the thermodynamic characteristics of the interaction also differ.     

High catalytic activity achieved with a mixed manganese-iron site in protein R2 of Chlamydia ribonucleotide reductase

Ribonucleotide reductase (class I) contains two components: protein R1 binds the substrate, and protein R2 normally has a diferric site and a tyrosyl free radical needed for catalysis. In Chlamydia trachomatis RNR, protein R2 functions without radical. Enzyme activity studies show that in addition to a diiron cluster, a mixed manganese-iron cluster provides the oxidation equivalent needed to initiate catalysis. An EPR signal was observed from an antiferromagnetically coupled high-spin Mn(III)-Fe(III) cluster in a catalytic reaction mixture with added inhibitor hydroxyurea. The manganese-iron cluster in protein R2 confers much higher specific activity than the diiron cluster does to the enzyme.     

The membrane ATPase of Escherichia coli I. Ion dependence and ATP-ADP exchange reaction

The properties of the membrane-bound ATPase (EC 3.6.1.3) of Escherichia coli have been reexamined using membranes obtained by mechanical disruption of exponentially growing cells.

The activity exhibited an absolute requirement for Mg²⁺ in the neutral pH range, while Ca²⁺ was found able to activate ATPase at more alkaline pH. Optimal activity was observed at pH 7.5, with a Mg/ATP ratio of 0.5.

ADP was found to inhibit ATP hydrolysis and to transform the Michaelian ATP concentration dependence with a K_m of 0.5 mM into a sigmoid curve with increasing K_m and decreasing V.

In contrast ADP activated an ATP-ADP exchange process and this shift from hydrolysis to exchange was stimulated by high Mg²⁺ and by orthophosphate.

All nucleoside triphosphates tested interfered with ATP hydrolysis, all could be hydrolyzed and could donate their terminal phosphate group to ADP. The relative efficiencies of nucleoside triphosphates in these three processes varied in parallel with minor discrepancies.

ATP hydrolysis was inhibited by N,N′-dicyclohexylcarbodiimide (DCCD) Dio 9, NaN₃ and pyrophosphate, the first two being ineffective against ATP-ADP exchange, the third being stimulatory and the last inhibitory.

ATP hydrolysis and ATP-ADP exchange are tentatively attributed to the terminal enzyme of oxidative phosphorylation.     

The pur3 gene from the pur cluster encodes a monophosphatase essential for puromycin biosynthesis in Streptomyces

The pur3 gene of the puromycin (pur) cluster from Streptomyces alboniger is essential for the biosynthesis of this antibiotic. Cell extracts from Streptomyces lividans containing pur3 had monophosphatase activity versus a variety of mononucleotides including 3'-amino-3'-dAMP (3'-N-3'-dAMP), (N6,N6)-dimethyl-3'-amino-3'-dAMP (PAN-5'-P) and AMP. This is in accordance with the high similarity of this protein to inositol monophosphatases from different sources. Pur3 was expressed in Escherichia coli as a recombinant protein and purified to apparent homogeneity. Similar to the intact protein in S. lividans, this recombinant enzyme dephosphorylated a wide variety of substrates for which the lowest Km values were obtained for the putative intermediates of the puromycin biosynthetic pathway 3'-N-3'-dAMP (Km = 1.37 mM) and PAN-5'-P (Km = 1.40 mM). The identification of this activity has allowed the revision of a previous proposal for the puromycin biosynthetic pathway.     

Oxidoreduction of cytochrome b in the presence of antimycin

1. The effect of oxidizing equivalents on the redox state of cytochrome b in the presence of antimycin has been studied in the presence and absence of various redox mediators.

2. The antimycin-induced extra reduction of cytochrome b is always dependent on the initial presence of an oxidant such as oxygen. After removal of the oxidant this effect remains or is partially (under some conditions even completely) abolished depending on the redox potential of the substrate used and the leak through the antimycin-inhibited site.

3. The increased reduction of cytochrome b induced by oxidant in the presence of antimycin involves all three spectroscopically resolvable b components (b-562, b-566 and b-558.

4. Redox mediators with an actual redox potential of less than 100–170 mV cause the oxidation of cytochrome b reduced under the influence of antimycin and oxidant.

5. Redox titrations of cytochrome b with the succinate/fumarate couple were performed aerobically in the presence of cyanide. In the presence of antimycin two b components are separated potentiometrically, one with an apparent midpoint potential above 80 mV (at pH 7.0), outside the range of the succinate/fumurate couple, and one with an apparent midpoint potential of 40 mV and an n value of 2. In the absence of antimycin cytochrome b titrates essentially as one species with a midpoint potential of 39 mV (at pH 7.0) and n = 1.14.

6. The increased reducibility of cytochrome b induced by antimycin plus oxidant is considered to be the result of two effects: inhibition of oxidation of ferrocytochrome b by ferricytochrome c₁ (the effect of antimycin), and oxidation of the semiquinone form of a two-equivalent redox couple such as ubiquinone/ubiquinol by the added oxidant, leading to a decreased redox potential of the QH₂/QH^• couple and reduction of cytochrome b.     

Effects of isocoumarins isolated from Paepalanthus bromelioides on mitochondria: uncoupling, and induction/inhibition of mitochondrial permeability transition

Isolated mitochondria may undergo uncoupling, and in presence of Ca(2+) at different conditions, a mitochondrial permeability transition (MPT) linked to protein thiol oxidation, and demonstrated by CsA-sensitive mitochondrial swelling; these processes may cause cell death either by necrosis or by apoptosis. Isocoumarins isolated from the Brazilian plant Paepalanthus bromelioides (Eriocaulaceae) paepalantine (9,10-dihydroxy-5,7-dimethoxy-1H-naptho(2,3c)pyran-1-one), 8,8'-paepalantine dimer, and vioxanthin were assayed at 1-50 microM on isolated rat liver mitochondria, for respiration, MPT, protein thiol oxidation, and interaction with the mitochondrial membrane using 1,6-diphenyl-1,3,5-hexatriene (DPH). The isocoumarins did not significantly affect state 3 respiration of succinate-energized mitochondria; they did however, stimulate 4 respiration, indicating mitochondrial uncoupling. Induction of MPT and protein thiol oxidation were assessed in succinate-energized mitochondria exposed to 10 microM Ca(2+); inhibition of these processes was assessed in non-energized organelles in the presence of 300 microM t-butyl hydroperoxide plus 500 microM Ca(2+). Only paepalantine was an effective MPT/protein thiol oxidation inducer, also releasing cytochrome c from mitochondria; the protein thiol oxidation, unlike mitochondrial swelling, was neither inhibited by CsA nor dependent on the presence of Ca(2+). Vioxanthin was an effective inhibitor of MPT/protein thiol oxidation. All isocoumarins inserted deeply into the mitochondrial membrane, but only paepalantine dimer and vioxantin decreased the membrane's fluidity. A direct reaction with mitochondrial membrane protein thiols, involving an oxidation of these groups, is proposed to account for MPT induction by paepalantine, while a restriction of oxidation of these same thiol groups imposed by the decrease of membrane fluidity, is proposed to account for MPT inhibition by vioxanthin.     

Effect of sydnone SYD-1, a mesoionic compound, on energy-linked functions of rat liver mitochondria

An important antitumour effect of SYD-1 (3-[4-chloro-3-nitrophenyl]-1,2,3-oxadiazolium-5-olate) has been shown. We now report the effects of this mesoionic compound on mitochondrial metabolism. SYD-1 (1.5 micromol mg(-1) protein) dose-dependently inhibited the respiratory rate by 65% and 40% in state 3 using sodium glutamate and succinate, respectively, as substrates. Phosphorylation efficiency was depressed by SYD-1, as evidenced by stimulation of the state 4 respiratory rate, which was more accentuated with glutamate ( approximately 180%) than with succinate ( approximately 40%), with 1.5 micromol mg(-1) protein of SYD-1. As a consequence of the effects on states 3 and 4, the RCC and ADP/O ratios were lowered by SYD-1 using both substrates, although this effect was stronger with glutamate. The formation of membrane electrical potential was inhibited by approximately 50% (1.5 micromol SYD-1mg(-1) protein). SYD-1 interfered with the permeability of the inner mitochondrial membrane, as demonstrated by assays of mitochondrial swelling in the presence of sodium acetate and valinomycin +K(+). SYD-1 (1.5 micromol mg(-1) protein) inhibited glutamate completely and succinate energized-mitochondrial swelling by 80% in preparations containing sodium acetate. The swelling of de-energized mitochondria induced by K(+) and valinomycin was inhibited by 20% at all concentrations of SYD-1. An analysis of the segments of the respiratory chain suggested that the SYD-1 inhibition site goes beyond the complex I and includes complexes III and IV. Glutamate dehydrogenase was inhibited by 20% with SYD-1 (1.5 micromol mg(-1) protein). The hydrolytic activity of complex F(1)F(o) ATPase in intact mitochondria was greatly increased ( approximately 450%) in the presence of SYD-1. Our results show that SYD-1 depresses the efficiency of electron transport and oxidative phosphorylation, suggesting that these effects may be involved in its antitumoural effect.     

Tau protein binds single-stranded DNA sequence specifically--the proof obtained in vitro with non-equilibrium capillary electrophoresis of equilibrium mixtures

Tau is a microtubule-associated protein, which plays an important role in physiology and pathology of neurons. Tau has been recently reported to bind double-stranded DNA (dsDNA) but not to bind single-stranded DNA (ssDNA) [Cell. Mol. Life Sci. 2003, 60, 413-421]. Here, we prove that tau binds not only dsDNA but also ssDNA. This finding was facilitated by using two kinetic capillary electrophoresis methods: (i) non-equilibrium capillary electrophoresis of equilibrium mixtures (NECEEM); (ii) affinity-mediated NECEEM. Using the new approach, we observed, for the first time, that tau could induce dissociation of strands in dsDNA by binding one of them in a sequence-specific fashion. Moreover, we determined the equilibrium dissociation constants for all tau-DNA complexes studied.     

The involvement of caspase-11 in TPEN-induced apoptosis

The depletion of intracellular zinc with N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN) induces protein synthesis-dependent apoptosis. Here we examined the involvement of caspase induction in apoptosis. Among the examined caspases, only caspase-11 was increased by TPEN. Caspase-11 activity also increased, which resulted in caspase-3 activation. Cycloheximide or actinomycin D blocked caspase-11 induction, reduced caspase-11 and -3 activation, and attenuated TPEN-induced neuronal apoptosis. Blockade of caspase-11 by a chemical inhibitor or genetic deletion attenuated TPEN-induced apoptosis, indicating a critical role of caspase-11 in TPEN-induced apoptosis. Although mitochondria-mediated caspase-9/-3 activation also contributed to TPEN-induced apoptosis, caspase-11 is likely a key inducible apoptosis-inducing protein.     

PAC-1 Activates Procaspase-3 in Vitro through Relief of Zinc-Mediated Inhibition

The direct induction of apoptosis has emerged as a powerful anticancer strategy, and small molecules that either inhibit or activate certain proteins in the apoptotic pathway have great potential as novel chemotherapeutic agents. Central to apoptosis is the activation of the zymogen procaspase-3 to caspase-3. Caspase-3 is the key “executioner” caspase, catalyzing the hydrolysis of a multitude of protein substrates within the cell. Interestingly, procaspase-3 levels are often elevated in cancer cells, suggesting a compound that directly stimulates the activation of procaspase-3 to caspase-3 could selectively induce apoptosis in cancer cells. We recently reported the discovery of a compound, PAC-1, which enhances procaspase-3 activity in vitro and induces apoptotic death in cancer cells in culture and in mouse xenograft models. Described herein is the mechanism by which PAC-1 activates procaspase-3 in vitro. We show that zinc inhibits the enzymatic activity of procaspase-3 and that PAC-1 strongly activates procaspase-3 in buffers that contain zinc. PAC-1 and zinc form a tight complex with one another, with a dissociation constant of approximately 42 nM. The combined data indicate that PAC-1 activates procaspase-3 in vitro by sequestering inhibitory zinc ions, thus allowing procaspase-3 to autoactivate itself to caspase-3. The small-molecule-mediated activation of procaspases has great therapeutic potential and thus this discovery of the in vitro mechanism of action of PAC-1 is critical to the development and optimization of other procaspase-activating compounds.     

Identification of non-specific lipid transfer protein-1 as a calmodulin-binding protein in Arabidopsis

Although non-specific lipid transfer proteins (nsLTPs) are widely present in plants, their functions and regulations have not been fully understood. In this report, Arabidopsis nsLTP1 was cloned and expressed to investigate its binding to calmodulin (CaM). Gel overlay assays revealed that recombinant nsLTP1 bound to CaM in a calcium-independent manner. The association of nsLTP1 and CaM was corroborated using CaM-Sepharose beads to specifically isolate recombinant nsLTP1 from crude bacterial lysate. The CaM-binding site was mapped in nsLTP1 to the region of 69-80 amino acids. This region is highly conserved among plant nsLTPs, implicating that nsLTPs are a new family of CaM-binding proteins whose functions may be mediated by CaM signaling.     

The goat mammary glandular epithelial (GMGE) cell line promotes polyfucosylation and N,N'-diacetyllactosediaminylation of N-glycans linked to recombinant human erythropoietin

We have established a continuous, non-transformed cell line from primary cultures from Capra hircus mammary gland. Low-density cultures showed a homogeneous epithelial morphology without detectable fibroblastic or myoepithelial cells. The culture was responsive to contact inhibition of proliferation and its doubling time was dependent on the presence of insulin and epidermal growth factor (EGF). GMGE cells secrete caseins regardless of the presence or absence of lactogenic hormones in the culture media. Investigation of the total N-glycan pool of human erythropoietin (rhEPO) expressed in GMGE cells by monosaccharide analysis, HPLC profiling, and mass spectrometry, indicated significant differences with respect to the same protein expressed in Chinese hamster ovary (CHO) cells. N-Glycans of rhEPO-GMGE are core-fucosylated, but fucosylation of outer arms was also found. Our results also revealed the presence of low levels of sialylation (>95% Neu5Ac), N,N'-diacetyllactosediamine units, and possibly Gal-Gal non-reducing terminal elements.     

Zinc in lipase L1 from Geobacillus stearothermophilus L1 and structural implications on thermal stability

Lipase L1 from Geobacillus stearothermophilus L1 contains an unusual extra domain, making a tight intramolecular interaction with the main catalytic domain through a Zn2+-binding coordination. To elucidate the role of the Zn2+, we disrupted the Zn2+-binding site by mutating the zinc-ligand residues (H87A, D61A/H87A, and D61A/H81A/H87A/D238A). The activity vs. temperature profiles of the mutant enzymes showed that the disruption of the Zn2+-binding site resulted in a notable decrease in the optimal temperature for maximal activity from 60 to 45-50 degrees C. The mutations also abolished the Zn2+-induced thermal stabilization. The wild-type enzyme revealed a 34.6-fold increase in stabilization with the addition of Zn2+ at 60 degrees C, whereas the mutant enzymes exhibited no response to Zn2+. Additional circular dichroism spectroscopy studies also confirmed the structural stabilizing role of Zn2+ on lipase L1 at elevated temperatures.     

Antioxidant activity of isocoumarins isolated from Paepalanthus bromelioides on mitochondria

The isocoumarins (1-50 microM) paepalantine (9,10-dihydroxy-5,7-dimethoxy-1H-naptho(2,3c)pyran-1-one), 8,8'-paepalantine dimer, and vioxanthin isolated from Paepalanthus bromelioides, were assessed for antioxidant activity using isolated rat liver mitochondria and non-mitochondrial systems, and compared with the flavonoid quercetin. The paepalantine and paepalantine dimers, but not vioxanthin, were effective at scavenging both 1,1-diphenyl-2-picrylhydrazyl (DPPH(*)) and superoxide (O(2)(-)) radicals in non-mitochondrial systems, and protected mitochondria from tert-butylhydroperoxide-induced H(2)O(2) accumulation and Fe(2+)-citrate-mediated mitochondrial membrane lipid peroxidation, with almost the same potency as quercetin. These results point towards paepalantine, followed by paepalantine dimer, as being a powerful agent affording protection, apparently via O(2)(-) scavenging, from oxidative stress conditions imposed on mitochondria, the main intracellular source and target of those reactive oxygen species. This strong antioxidant action of paepalantine was reproduced in HepG2 cells exposed to oxidative stress condition induced by H(2)O(2).     

Convergence in pigmentation at multiple levels: mutations,genes and function

Convergence—the independent evolution of the same trait by two or more taxa—has long been of interest to evolutionary biologists, but only recently has the molecular basis of phenotypic convergence been identified. Here, we highlight studies of rapid evolution of cryptic coloration in vertebrates to demonstrate that phenotypic convergence can occur at multiple levels: mutations, genes and gene function. We first show that different genes can be responsible for convergent phenotypes even among closely related populations, for example, in the pale beach mice inhabiting Florida''s Gulf and Atlantic coasts. By contrast, the exact same mutation can create similar phenotypes in distantly related species such as mice and mammoths. Next, we show that different mutations in the same gene need not be functionally equivalent to produce similar phenotypes. For example, separate mutations produce divergent protein function but convergent pale coloration in two lizard species. Similarly, mutations that alter the expression of a gene in different ways can, nevertheless, result in similar phenotypes, as demonstrated by sister species of deer mice. Together these studies underscore the importance of identifying not only the genes, but also the precise mutations and their effects on protein function, that contribute to adaptation and highlight how convergence can occur at different genetic levels.     

Puroindolines form ion channels in biological membranes

Wheat seeds contain different lipid binding proteins that are low molecular mass, basic and cystine-rich proteins. Among them, the recently characterized puroindolines have been shown to inhibit the growth of fungi in vitro and to enhance the fungal resistance of plants. Experimental data, using lipid vesicles, suggest that this antimicrobial activity is related to interactions with cellular membranes, but the underlying mechanisms are still unknown. This paper shows that extracellular application of puroindolines on voltage-clamped Xenopus laevis oocytes induced membrane permeabilization. Electrophysiological experiments, on oocytes and artificial planar lipid bilayers, suggest the formation, modulated by voltage, of cation channels with the following selectivity: Cs(+) > K(+) > Na(+) > Li(+) > choline = TEA. Furthermore, this channel activity was prevented by addition of Ca(2+) ions in the medium. Puroindolines were also able to decrease the long-term oocyte viability in a voltage-dependent manner. Taken together, these results indicate that channel formation is one of the mechanisms by which puroindolines exert their antimicrobial activity. Modulation of channel formation by voltage, Ca(2+), and lipids could introduce some selectivity in the action of puroindolines on natural membranes.     

The effect of calcium ions on subcellular localization of aldolase-FBPase complex in skeletal muscle

In skeletal muscles, FBPase-aldolase complex is located on alpha-actinin of the Z-line. In the present paper, we show evidence that stability of the complex is regulated by calcium ions. Real time interaction analysis, confocal microscopy and the protein exchange method have revealed that elevated calcium concentration decreases association constant of FBPase-aldolase and FBPase-alpha-actinin complex, causes fast dissociation of FBPase from the Z-line and slow accumulation of aldolase within the I-band and M-line. Therefore, the release of Ca2+ during muscle contraction might result, simultaneously, in the inhibition of glyconeogenesis and in the acceleration of glycolysis.     

Identification and characterization of PorH, a new cell wall channel of Corynebacterium glutamicum

The cell wall of Corynebacterium glutamicum contains the cation-selective channel (porin) PorA_C.glut and the anion-selective channel PorB_C.glut for the passage of hydrophilic solutes. Lipid bilayer experiments with organic solvent extracts of whole C. glutamicum cells cultivated in minimal medium suggested that also another cation-selective channel-forming protein, named PorH_C.glut, is present in C. glutamicum. The protein was purified to homogeneity by fast-protein liquid chromatography across a HiTrap-Q column. The pure protein had an apparent molecular mass of about 12 kDa on SDS-PAGE. Western blot analysis suggested that the cell wall channel is presumably formed by protein oligomers. The purified protein forms cation-selective channels with an average single-channel conductance of about 2.5 nS in 1 M KCl in the lipid bilayer assay. The PorH_C.glut protein was partially sequenced, and based on the resulting amino acid sequence, the corresponding gene, designated as porH_C.glut, was identified in the published genome sequence of C. glutamicum ATCC13032. PorH_C.glut contains only the inducer methionine but no N-terminal extension, which suggests that the export and assembly of the protein follow a yet unknown pathway. PorH_C.glut is coded in the bacterial chromosome by a gene that is localized in the vicinity of porA_C.glut, within a putative operon of 13 genes. RT-PCR revealed that both porins are cotranscribed. They coexist according to immunological detection experiments in the cell wall of C. glutamicum together with PorB_C.glut and PorC_C.glut.     

Divergence of the mitochondrial electron transport chains from the green alga Chlamydomonas reinhardtii and its colorless close relative Polytomella sp.

Compelling evidence exists that the colorless algae of the genus Polytomella arose from a green Chlamydomonas-like ancestor by losing its functional photosynthetic apparatus. Due to the close relationship between the colorless and the green chlorophyte, Polytomella sp. appeared as a useful indicative framework for structural studies of Chlamydomonas reinhardtii mitochondria. However, comparative studies reported here unexpectedly revealed significant differences between the mitochondrial respiratory systems of the two algae. Two-dimensional blue native/SDS-PAGE of isolated mitochondria indicated that cytochrome-containing respiratory complexes III and IV in the two chlorophytes contrast in size, subunit composition and relative abundance. Complex IV in Polytomella is smaller than its counterpart in C. reinhardtii and occurs in two forms that differ presumably in the presence of subunit COXIII. The cytochrome c and the iron-sulfur Rieske protein of both chlorophytes revealed structural differences on the amino acid sequence level. Under comparable culture conditions, the colorless alga exhibits lower levels of cytochrome c and complex IV but a higher respiratory activity than the green alga. Cytochrome c levels were also found to be differently regulated by the growth conditions in both algae. The divergence between the respiratory systems in the two related chlorophytes can be viewed as a consequence of the loss of photosynthetic activity and/or of the adaptation to the environment via the acquisition of a more flexible, heterotrophic metabolism. Our understanding of mitochondrial function and evolution is expected to be greatly enhanced via further parallel studies of photosynthetic/non-photosynthetic algae, for which this study forms an incentive.     

High affinity selenium uptake in a keratinocyte model

The distribution of selenium in mammals has been recently shown to be mediated primarily by selenoprotein P. Even in the absence of selenoprotein P, selenium is distributed from the liver into all organs and tissues when supplemented in the diet. The form of selenium that is actively taken up by mammalian cells at trace concentrations has yet to be determined. We used a human keratinocyte model to determine whether reduction of the oxyanion selenite (SeO(3)(2-)) to the more reduced form of selenide (HSe(-)) would affect uptake. Indeed a reduced form of selenium, presumably selenide, was actively transported into keratinocytes and displayed saturation kinetics with an apparent K(m) of 279 nM. ATPase inhibitors blocked the uptake of selenide, as did the competing anions molybdate and chromate, but not sulfate. These results suggest that the small molecule form of selenium that is distributed in tissues is hydrogen selenide, despite its sensitivity to oxygen and reactivity to thiols.